Lantibiotics are a group of ribosomally synthesized peptide antibiotics that are post-translationally modified by multi- enzyme complexes to their bioactive forms. The recent cloning of the gene clusters involved in the biosynthesis of many lantibiotics has laid the foundation for genetic engineering aimed at in vivo production of novel compounds with potentially interesting properties. These studies revealed that the enzymes responsible for these: processes lack absolute substrate specificity. Genetic protein engineering is limited to the 20 physiological amino acids. However, since the size of the pre- peptides of lantibiotics are well within the limit of solid phase peptide synthesis, the pool of available amino acids that can be used for "chemical protein engineering" is increased dramatically. Thus, protein engineering using chemically synthesized unnatural peptide substrates may provide a valuable tool to study structure-function relationships in lantibiotic biosynthesis, and to establish their molecular mechanism(s) of cytotoxicity. In order to achieve these goals, high level heterologous expression systems for the proteins involved in the biosynthesis of the lantibiotics epidermin and subtilin will be developed. Furthermore, synthetic methodology to synthesize analogs of the pre-peptides will be investigated. The synthetic peptides will be used to explore the stereochemical and structural requirements for successful post-translational modification. Once these requirements have been defined, this methodology may be used for the generation of lantibiotics with more potent antibacterial activity. Finally, this strategy also allows the site-specific introduction into lantibiotics of radiolabels, fluorescent or photoaffinity probes, and spin labels, providing valuable tools for the identification of the biological targets of lantibiotics, and to evaluate the molecular basis of their cytotoxicity.